PROJECT SUMMARY/ABSTRACT Mechanical forces are central to a large number of cellular processes, such as cell division, cell motility, organelle morphogenesis and stem cell differentiation, to name a few. Forces are produced by molecular machineries that convert biochemical energy into mechanical energy. Despite the ubiquity of forces in cells, little is known about the molecular mechanisms of force production and force transmission in vivo, essentially, because there is a lack of universal tools and methods to directly measure forces applied at the molecular level in live cells. This project aims to develop new universally applicable molecular force sensors to measure the magnitude of forces produced on individual proteins in vivo. To create these new force sensors we will rationally design coiled-coils (CCs) that unzip under specific forces between ~1 to ~15 piconewtons (aim 1). We will calibrate select CCs using optical tweezers (aim 2). To measure forces on a protein of interest (POI) in vivo, we will create a library of strains where individual CCs are inserted between key domains of the POI. If the force applied on the POI chimera is larger than the force to unzip the CC, the protein will lose functionality. By determining the functionality of each chimeric construct, we will be able to determine the forces applied on the POI by dichotomy. As a proof of principle, we will use this strategy to measure the forces on two cytoskeletal proteins involved in clathrin-mediated endocytosis in fission yeast (aim 3). If successful, our strategy has the potential to highly impact and transform the study of mechanisms of force production, force sensing and mechanotransduction in virtually all life forms, and all cellular and developmental processes.